Supramolecular complexes as photoactivated DNA cleavage agents

ABSTRACT

The invention provides supramolecular metal complexes as DNA cleaving agents. In the complexes, charge is transferred from one light absorbing metal (e.g. Ru or Os) to an electron accepting metal (e.g. Rh) via a bridging π-acceptor ligand. A bioactive metal-to-metal charge transfer state capable of cleaving DNA is thus generated. The complexes function when irradiated with low energy visible light with or without molecular oxygen.

This invention was made using funds from a grant from the NationalScience Foundation having grant number CHE-9632713. The government mayhave certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention generally relates to photodynamic therapy agents. Inparticular, the invention provides tunable supramolecular metalliccomplexes which can be activated to cleave DNA by low energy light andin the absence of O₂.

2. Background of the Invention

Photodynamic therapy (PDT) is currently gaining acceptance for thetreatment of hyperproliferating tissues such as cancers andnon-malignant lesions. Significant emphasis has been placed ondeveloping photochemical reagents capable of cleaving DNA for suchpurposes. Photochemical approaches are of particular interest as theyoffer reaction control and can be highly targeted.

One popular approach in the design of photodynamic agents involves thesensitization of molecular oxygen. Typically, such agents absorb lightenergy and transfer that energy to molecular oxygen to generate areactive singlet oxygen state ¹O₂. The ¹O₂ state is highly reactive and,in an intracellular environment, ¹O₂ randomly reacts with and damagesbiomolecules and subcellular components, leading to potentially lethaldamage to the cell. However, the use of such agents has severaldrawbacks. For example, the wavelengths of light that must be used toactivate this type of photodynamic agent are short wavelength/highenergy and cause extensive damage to healthy tissue adjacent to thetargeted, hyperproliferating cells. The targeted cell may lyse,releasing ¹O₂ into the immediate environment where it continues to reactrandomly with and damage healthy tissue in the area. Further, suchagents require the presence of oxygen, the level of which is relativelylow in an intracellular environment. Finally, there is an overall lackof flexibility in the design of such agents.

It is thus of interest to develop photosensitizing agents forphotodynamic therapy with alternative mechanisms of action. Inparticular, it would be of benefit to have available photosensitizingagents that absorb and are activated by low energy light. The use ofphotosensitizing agents that absorb low energy light is less likely tocause unwanted collateral damage to non-targeted cells in a photodynamictherapy setting. In addition, it would be of benefit to have availablephotosensitizing agents that function efficiently in the absence ofmolecular oxygen as such agents would be particularly suitable forintracelluar use. Further, it would be highly desirable to haveavailable tunable photosensitizing agents, i.e. photosensitizing agentswith a flexible architectural motif that can be readily adjusted ortailored for use in specific applications.

SUMMARY OF THE INVENTION

The present invention provides novel metal-based DNA cleaving agents.The agents are supramolecular metallic complexes containing at least onemetal to ligand charge transfer (MLCT) light absorbing metal, at leastone bridging T-acceptor ligand, and an electron acceptor metal. Thecomplexes are capable of effecting the cleavage of DNA upon exposure tolow energy visible light, and do so in the absence of oxygen.

The invention provides new compositions of matter of the forms:

1)[(2,2′-bipyridine)₂Os(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)Os(2,2′-bipyridine)₂](X)₅, where X is a counterion selected from thegroup consisting of PF₆ ⁻, Cl⁻, Br⁻, CF₃SO₃ ⁻ and BF₄ ⁻.

2)[(2,2′:6′,2″-terpyridine)RuCl(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)RuCl(2,2′:6′,2″-terpyridine)](X)₃, where X is a counterionselected from the group consisting of PF₆ ⁻, Cl⁻, Br⁻, CF₃SO₃ ⁻ and BF₄⁻; and

3)[(2,2′:6′,2″-terpyridine)RuCl(2,2′-bipyridimidine)RhCl₂(2,2′-bipyridimidine)RuCl(2,2′:6′,2″-terpyridine)](X)₃,where X is a counterion selected from the group consisting of PF₆ ⁻,Cl⁻, Br⁻, CF₃SO₃ ⁻ and BF₄ ⁻. The invention also provides composition,comprising at least one of the above compounds dissolved or dispersed ina carrier.

The invention further provides a method for cleaving DNA. The methodincludes the steps of combining the DNA with a supramolecular complex.The complex contains at least one metal to ligand charge transfer (MLCT)light absorbing metal, at least one bridging i-acceptor ligand, and anelectron acceptor metal. The step of combining is carried out underconditions that allow the supramolecular complex to bind to the DNA, andthe supramolecular complex is present in sufficient quantity to cleavesaid DNA. The second step of the method is exposing the DNA to light orradiant energy in an amount sufficient to activate the supramolecularcomplex to cleave the DNA. The metal to ligand charge transfer (MLCT)light absorbing metal may be, for example, ruthenium(II), osmium(III),rhenium(I), iron(II) or platinum(II). The bridging π-acceptor ligand maybe, for example, 2,3-bis(2-pyridyl)pyrazine; 2,2′-bipyridimidine;2,3-bis(2-pyridyl)quinoxaline; or 2,3,5,6,-tetrakis(2-pyridyl)pyrazine.The electron acceptor metal may be, for example, rhodium(III),platinum(IV), cobalt(III), or iridium(III). The supramolecular complexmay further include at least one terminal π-acceptor ligand, in whichcase the terminal π-acceptor ligand may be, for example,2,2′-bipyridine; 2,2′:6′,2″-terpyridine; triphenylphosphine; and2,2′-phenylpyridine or diethylphenylphosphine. In a preferredembodiment, the light used to activate the complex is visible light.

The supramolecular complex utilized in the method may be, for example,[(2,2′-bipyridine)₂Ru(2-pyridyl)pyrazine)RhCl₂(2-pyridyl)pyrazine)Ru(2,2′-bipyridine)₂](PF₆)₅;[(2,2′-bipyridine)₂Os(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)Os(2,2′-bipyridine)₂](PF₆)₅;[(2,2′:6′,2″-terpyridine)RuCl(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)RuCl(2,2′:6′,2″-terpyridine)](PF₆)₃; or[(2,2′:6′,2″-terpyridine)RuCl(2,2′-bipyridimidine)RhCl₂(2,2′-bipyridimidine)RuCl(2,2′:6′,2″-terpyridine)](PF₆)₃′In addition, the combining step of the method may occur within ahyperproliferating cell.

The invention also provides a composition for effecting the cleavage ofDNA in hyperproliferating cells. The composition contains asupramolecular complex comprising at least one metal to ligand chargetransfer (MLCT) light absorbing metal; at least one bridging n-acceptorligand; and an electron acceptor metal. The metal to ligand chargetransfer (MLCT) light absorbing metal may be ruthenium(II), osmium(III),rhenium(I), iron(II) or platinum(II). The bridging π-acceptor ligand maybe 2,3-bis(2-pyridyl)pyrazine; 2,2′-bipyridimidine; 2,3-bis(2-pyridyl)quinoxaline; or 2,3,5,6,-tetrakis(2-pyridyl)pyrazine. The electronacceptor metal may be rhodium(III), platinum(IV), cobalt(III), oriridium(III). The supramolecular complex may further comprises at leastone terminal π-acceptor ligand such as 2,2′-bipyridine;2,2′:6′,2″-terpyridine; triphenylphosphine; and 2,2′-phenylpyridine ordiethylphenylphosphine. The supromolecular complex may be dissolved ordispersed in a carrier.

The supramolecular complex in the composition may be [(2,2′-bipyridine)₂Ru(2-pyridyl)pyrazine)RhCl₂(2-pyridyl)pyrazine)Ru(2,2′-bipyridine)₂](PF₆)₅;[(2,2′-bipyridine)₂Os(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)Os(2,2′-bipyridine)₂](PF₆)₅;[(2,2′:6′,2″-terpyridine)RuCl(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)RuCl(2,2′:6′,2″-terpyridine)](PF₆)₃;or[(2,2′:6′,2″-terpyridine)RuCl(2,2′-bipyridimidine)RhCl₂(2,2′-bipyridimidine)RuCl(2,2′:6′,2″-terpyridine)](PF₆)₃.

The invention further provides a method for decreasing the replicationof hyperproliferating cells. The method includes the steps of deliveringa supramolecular complex to the cells, the complex containing at leastone metal to ligand charge transfer (MLCT) light absorbing metal; atleast one bridging π-acceptor ligand; and an electron acceptor metal.The method further includes the step of applying light or radiant energyto the hyperproliferating cells. The step of applying light to thehyperproliferating cells induces the production of a metal-to-metalcharge transfer state within the supramolecular complex. Themetal-to-metal charge transfer state mediates the cleavage of DNA of thehyperproliferating cells, thereby causing a decrease in the replicationof the hyperproliferating cells.

In the method, the at least one metal to ligand charge transfer (MLCT)light absorbing metal may be ruthenium(II), osmium(III), rhenium(I),iron(II) or platinum(II). The at least one bridging π-acceptor ligandmay be 2,3-bis(2-pyridyl)pyrazine; 2,2′-bipyridimidine;2,3-bis(2-pyridyl)quinoxaline; or 2,3,5,6,-tetrakis(2-pyridyl)pyrazine.The electron acceptor metal may be rhodium(III), platinum(IV),cobalt(III), or iridium(III). The supramolecular complex may furthercomprises at least one terminal n-acceptor ligand such as2,2′-bipyridine; 2,2′:6′,2″-terpyridine; triphenylphosphine; and2,2′-phenylpyridine and diethylphenylphosphine. The light may be visiblelight. The supramolecular complex may be [(2,2′-bipyridine)₂Ru(2-pyridyl)pyrazine)RhCl₂(2-pyridyl)pyrazine)Ru(2,2′-bipyridine)₂](PF₆)₅;[(2,2′-bipyridine)₂Os(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)Os(2,2′-bipyridine)₂](PF₆)₅;[(2,2′:6′,2″-terpyridine)RuCl(2,3-bis(2-pyridyl)pyrazine)RhCl₂(2,3-bis(2-pyridyl)pyrazine)RuCl(2,2′:6′,2″-terpyridine)](PF₆)₃;or[(2,2′:6′,2″-terpyridine)RuCl(2,2′-bipyridimidine)RhCl₂(2,2′-bipyridimidine)RuCl(2,2′:6′,2″-terpyridine)](PF₆)₃. The hyperproliferating cells may becancer cells.

The invention further provides a method for decreasing the replicationof hyperproliferating cells. The method comprises the steps ofdelivering to said hyperproliferating cells a supramolecular complexwhich contains at least one metal to ligand charge transfer (MLCT) lightabsorbing metal; at least one bridging π-acceptor ligand; and anelectron acceptor metal, followed by the step of inducing the productionof a metal-to-metal charge transfer state within the supramolecularcomplex by the application of light, thereby causing a decrease inreplication of the hyperproliferating cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-C. Molecular structure of A, dpp; B, bpm; and C, tpy.

FIG. 2. Schematic of building-block synthesis of[{(tpy)RuCl(bpm)}₂RhCl₂]³⁺.

FIGS. 3A and B. A, Orbital Energy Diagram for [{(tpy)RuCl(bpm)}₂RhCl₂]³⁺and [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺; B, Orbital Energy Diagram for[{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ and [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺.

FIGS. 4A and B. Cyclic voltammograms of the trimetallic complexes[{(tpy)-RuCl(BL)}₂RhCl₂](PF₆)₃ in 0.4 M Bu₄NPF₆ in CH₃CN, whereBL=2,3-bis(2-pyridyl)pyrazine, dpp (A), or 2,2′-bipyrimidine, bpm (B),and tpy=2,2′:6′,2″-terpyridine. Potentials recorded vs Ag/AgCl referenceelectrode (0.29 V vs NHE).

FIG. 5. Electrochemical Mechanism for the Ru, Rh, Ru Triads.

FIGS. 6A and B. A, Spectroelectrochemistry for[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃ where tpy=_(—)2,2′:6′,2″-terpyridine anddpp=2,3-bis(2-pyridyl)pyrazine in 0.1 M Bu₄NPF₆ in CH₃CN at roomtemperature: (−) [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺, ( . . . )[{(tpy)RuCl(dpp)}₂RhCl₂]⁵⁺. B, Spectroelectrochemistry for[{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃ where tpy=2,2′:6′,2″-terpyridine andbpm=2,2′-bipyrimidine in 0.1 M Bu₄NPF₆ in CH₃CN at room temperature: (−)[{(tpy)RuCl(bpm)}₂RhCl₂]³⁺, ( . . . )[{(tpy)RuCl(bpm)}₂RhCl₂]⁵⁺.

FIGS. 7A and B. Representations of the mixed-metal trimetallic complex[{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺.

FIG. 8. Electronic absorption spectra for [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ (s),[{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ ( - - - ), and [{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺ ( . .. ) in doubly distilled water, ddH₂O.

FIG. 9. Orbital Energy Diagram for [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ and[{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺

FIG. 10A-C. (a) Schematic representations of imaged agarose gel showingthe photocleavage of pUC18 plasmid by [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ in theabsence of molecular oxygen. Lane 1 λ molecular weight standard, lanes 2and 3 plasmid controls, lanes 4 and 6 plasmid incubated at 37° C. (2 h)in the presence of [(bpy)₂Ru(dpp)]²⁺ and [I{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺,respectively (1:5-metal complex/base pair), lanes 5 and 7 plasmidirradiated at λ≧475 nm for 10 min in the presence of [(bpy)₂Ru(dpp)]²⁺and [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺, respectively. (b) Lanes 1 and 2 plasmidcontrols, lanes 3 and 5 plasmid incubated at 37° C. (3 h) in thepresence of [{(bpy)₂Ru(bpm)} 2RhCl₂]⁵⁺ and [{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺,respectively, lanes 4 and 6 plasmid irradiated at λ≧475 nm for 10 min inthe presence of [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ and [{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺,respectively. (c) Imaged agarose gel showing photocleavage ofpBluescript plasmid in the absence of molecular oxygen by[(bpy)₂Ru(dpp)RhCl₂(dpp)Ru(bpy)₂](PF₆)₅. Lane 1 is the λ molecularweight standard, lane 2 is the control linearized DNA (cut with HindIII)with no metal present, lane 3 is the control circular DNA with no metalpresent, lane 4 is a 1:5 metal complex/base pair mixture of the plasmidwith the metal complex incubated at 37° C. (4 h), and lane 5 is a 1:5metal complex/base pair mixture of the plasmid with the metal complexphotolyzed at 520±5 nm for 4 h. All gels used 0.8% agarose, 90 mM Tris,and 90 mM boric acid buffer (pH=8.2, ionic strength=0.0043 M calculatedusing the Henderson-Hasselbalch equation).

FIGS. 11A and B. A, Photochemically induced inhibition of cellreplication: time course. x axis=time post illumination in minutes; yaxis=relative cell growth. B, Photochemically induced inhibition of cellreplication: effect of varying concentrations of complex. xaxis=concentration of [{(bpy)₂Ru(dpp)}₂RhCl₂]Cl₅; y axis=relative cellgrowth.

FIG. 12A-C. DNA Photocleavage by Various Supramolecular MetallicComplexes. Schematic representation of analysis of cleavage patterns bygel electrophoresis.

A, DNA Photocleavage of pUC18 using [{(bpy)₂Os(dpp)}₂RhCl₂](PF₆)₅: Lane1 is the λ molecular weight standard, Lane 2 is a plasmid control, Lane3 is a 1:5 metal complex/base pair mixture of the plasmid with the metalcomplex incubated at 37° C. for 20 minutes, Lane 4 is a 1:5 metalcomplex/base pair mixture of the plasmid with the metal complexphotolyzed at >475 nm for 20 minutes.

B, DNA Photocleavage of pBluescript using[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃: Lane 1 is the λ molecular weightstandard, Lane 2 is a plasmid control, Lane 3 is a 1:5 metalcomplex/base pair mixture of the plasmid with the metal complexincubated at 37° C. for 20 minutes, Lane 4 is a 1:5 metal complex/basepair mixture of the plasmid with the metal complex photolyzed at >475 nmfor 20 minutes.

C, DNA Photocleavage of pUC18 using [{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃: Lane1 is the λ molecular weight standard, Lane 2 is a plasmid control, Lane3 is the plasmid alone photolyzed λ>475 nm for 15 mins, Lane 4 is a 1:5metal complex/base pair mixture of the plasmid with the metal complexincubated at 37° C. for 15 minutes, Lane 5 is a 1:5 metal complex/basepair mixture of the plasmid with the metal complex photolyzed at >475 nmfor 15 minutes.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides novel metallic DNA cleaving agents thatexhibit unique supramolecular architecture. The agents aresupramolecular metal complexes in which a novel type of excitation isused. In the complexes, charge is transferred from one metal to anotherto generate a metal-to-metal charge transfer state, a unique type ofexcited state for application to photoactivated DNA cleavage. Thecomplexes are able to cleave DNA as a direct result of the moleculardesign which localizes the highest occupied molecular orbital on atleast one charge transfer light absorbing metal center, and the lowestunoccupied molecular orbital on a bioactive electron accepting metalcenter. This general molecular architectural scheme allows greatflexibility in terms of the design of DNA cleaving complexes since manydifferent substances may function as components of the system. Further,in contrast to most known photodynamic therapy agents, the DNA cleavingagents of the present invention do not require oxygen to function. Theythus function efficiently in intracellular environments where O₂ levelsare low. Also, since these agents do not generate singlet oxygen,incidental damage to healthy tissue due to release of ¹O₂ to thesurrounding environment cannot occur. In addition, the complexes areactivated by the application of visible, low energy light, thusprecluding unwanted cellular damage (e.g. of healthy tissue) whichoccurs as a result of the use of high energy light.

In the agents, three essential components are coupled: 1) at least onemetal to ligand charge transfer (MLCT) light absorbing metal center; 2)a bridging r-acceptor ligand; and 3) an electron acceptor metal center.The function of the metal to ligand charge transfer light absorber is toproduce an initially optically populated metal to ligand charge transferstate. Requirements of the bridging n-acceptor ligand are that it mustcoordinate to both the light absorbing metal and the electron acceptormetal, and possess a π system capable of being involved in an initialmetal to ligand charge transfer excitation. The requirement for theelectron acceptor metal is that it bind to the bridging π-acceptorligand and be energetically capable of accepting an electron from theoptically populated MLCT state to produce the reactive metal to metalcharge transfer (MMCT) state. Without being bound by theory, it isbelieved that it is the MMCT state that functions to cleave the DNA towhich the complex is bound.

In one embodiment of the present invention, two metal to ligand chargetransfer light absorbers are utilized. However, those of skill in theart will recognize that only one MLCT light absorber need be present inthe complex of the present invention. Alternatively, more than two suchlight absorbers may be incorporated to produce the initially opticallypopulated metal to ligand charge transfer state. The exact number andtype of MLCT light absorbers used in the supramolecular metalliccomplexes of the present invention may vary, depending on severalfactors including but not limited to: the desired excitation wavelengthto be employed; the oxidation potential of interest for the metal basedhighest occupied molecular orbital; the required extinction coefficientfor the excitation wavelength; ease of synthesis of the complex; costand/or availability of components; and the like. Any suitable number ofMLCT light absorbers may be used so long as within the complex aninitial optically populated MLCT state is produced upon exposure tolight or radiant energy, and which can be relayed to a suitable bridgingligand for transfer to an electron acceptor metal. In preferredembodiments, the number of MLCT light absorbers will range from 1 toabout 14, and preferably from 1 to about 5, and more preferably from 1to about 3. In one embodiment of the invention, two MLCT light absorbersare utilized.

Those of skill in the art will recognize that many suitable metals existthat can function as MLCT light absorbers in the practice of the presentinvention. Examples include but are not limited to ruthenium(II),osmium(II), rhenium (I), iron(II), platinum(II), etc. In preferredembodiments, two ruthenium(II) or two osmium(II) centers are utilized.

The complexes of the present invention require the presence of at leastone bridging π-acceptor ligand capable of being involved in an initialmetal to ligand charge transfer excitation. By “bridging ligand” we meanthat, in the supramolecular complex, the π-acceptor ligand is located orpositioned (i.e. bonded, coordinated) between an MLCT light absorber andan electron acceptor metal. Further, if there is more than one MLCTlight absorber in the complex, the bridging π-acceptor ligands will bepositioned to attach each light absorbing unit to either another lightabsorbing unit or directly to the electron accepting metal center.

The π-acceptor ligands coordinate or bind to the metal centers via donoratoms. Those of skill in the art will recognize that many suitablesubstances exist which contain appropriate donor atoms and may thusfunction as π-acceptor ligands in the complexes of the presentinvention. These π-acceptor ligands fall into two categories, bridgingand terminal ligands. Bridging ligands serve to connect metal centersand thus bind to or coordinate two separate metal centers.

Terminal ligands bind or coordinate to only one metal center and serveto satisfy the needed coordination sphere for such metals and provide ameans to tune both light absorbing and redox properties of that metalcenter. For example, substances with: nitrogen donor atoms (e.g.pyridine- and pyridimidine-containing moieties such as 2,2′-bipyridine(“bpy”); 2,2′:6′,2″-terpyridine (“tpy”); 2,3-bis(2-pyridyl)pyrazine(“dpp”); and 2,2′-bipyridimidine (“bpm”); 2,3-bis(2-pyridyl)quinoxaline;2,3,5,6,-tetrakis(2-pyridyl)pyrazine; carbon and nitrogen donor atoms(e.g. 2,2′-phenylpyridine); phosphorus donor atoms (e.g.triphenylphosphine, diethylphenylphosphine); etc. In preferredembodiments of the present invention, the π-acceptor ligands are bpy,tpy, dpp and bpm.

Further, those of skill in the art will recognize that, depending on thenumber of available coordination sites on the metals to which theπ-acceptor ligands are coordinated, other extraneous ligands may also bepresent to complete the coordination sphere of the metal. Examples ofsuch ligands include but are not limited to halogens such as Cl and Br,COOH, CO, H₂O, CH₃CN, etc.

The electron acceptor metal is an essential component of this moleculardesign. Those of skill in the art will recognize that many metals may beused as the electron acceptor metal in the complexes of the presentinvention. Examples of suitable metals include but are not limited torhodium(III), platinum(IV), cobalt(III), iridium(III). Any metal thatcan bind to a bridging π-acceptor ligand and accept an electron from theoptically populated MLCT state to produce the reactive MMCT state may beutilized. In a preferred embodiment of the invention, the electronacceptor metal is rhodium(III). Further, the number of electron acceptormetal centers in the complex may also be varied. Multifunctional systemscould be designed that use many electron acceptor sites to enhance thefunctioning of the system by providing additional bioactive sites withina single molecular architecture.

In general, the supramolecular architecture of the complexes of thepresent invention can be varied by changing the identity and number ofcomponents of the complex. However, it is necessary to retain thecomponents in sufficiently close location and appropriate orientation toprovide the necessary electronic coupling. This coupling is necessary toallow for electron transfer from the initial π-acceptor ligand, thataccepts the charge in the initially populated metal to ligand chargetransfer state, to the electron accepting metal center to lead to theformation of the reactive metal to metal charge transfer state. It isalso important that component separation and orientation not allow forrapid relaxation of the reactive MMCT state, facilitated by rapid backelectron transfer. Those of skill in the art will recognize that theprecise distances between components and the orientation of thecomponents will vary from complex to complex, depending on the identityof complex substituents. However, in general the distances will beconfined to the multi-atomic or multi-angstrom scale.

Exemplary forms of the complexes of the invention contain two ruthenium-or osmiun-based light absorbers which are coupled to a biologicallyactive rhodium metal site. In these embodiments, the light absorbingmetal centers are occupied by Ru or Os, and the central electronacceptor metal site is occupied by Rh.

Preferred embodiments of the complexes include:

-   [(bpy)₂Ru(dpp)RhCl₂(dpp)Ru(bpy)₂](X)₅-   [(bpy)₂Os(dpp)RhCl₂(dpp)Os(bpy)₂](X)₅-   [(tpy)RuCl(bpm)RhCl₂(bpm)RuCl(tpy)](X)₃ and-   [(tpy)RuCl(bpm)RhCl₂(bpm)RuCl(tpy)](X)₃;    where X is a counterion such as PF₆ ⁻, Cl⁻, Br⁻, CF₃SO₃ ⁻, BF₄ ⁻,    CLO₄ ⁻, SO₄ ²⁻, etc. Those of skill in the art will recognize that    many such suitable counterions exist and may be utilized to form the    salt form of a complex without altering the fundamental properties    of the complex, other than its solubility.

The invention further provides new compositions of matter:

-   [(bpy)₂Os(dpp)RhCl₂(dpp)Os(bpy)₂](X)₅-   [(tpy)RuCl(bpm)RhCl₂(bpm)RuCl(tpy)](X)₃ and-   [(tpy)RuCl(bpm)RhCl₂(bpm)RuCl(tpy)](X)₃;    where X is a counterion such as PF₆ ⁻, Cl⁻, Br⁻, CF₃SO₃ ⁻, BF₄ ⁻,    CLO₄ ⁻, SO₄ ²⁻, etc. as above.

The DNA cleaving agents of the present invention may be used forcleavage of DNA in many settings, including but not limited to cleavageof purified or partially purified DNA in laboratory setting forinvestigational purposes; and for the cleavage of DNA within cells,either ex vivo or in vivo. For example, ex vivo uses include cleavage ofDNA in cultured cells for any reason, or of cells that have been removedfrom an individual with the intent of reintroducing the cells into theindividual (or another individual) after manipulation of the cells (e.g.purging of tumor cells, genetic engineering of the cells, etc.) and thelike. Examples of in vivo uses include the cleavage of DNA of cellswithin an organism, especially unwanted hyperproliferating cells such astumor or cancer cells (including but are not limited to leukemia cells,ovarian cancer cells, Burkitt's lymphoma cells, breast cancer cells,gastric cancer cells, testicular cancer cells, and the like), and cellsassociated with psoriasis, warts, macular degeneration and othernon-malignant hyperproliferating conditions.

While the method of the present invention is principally intended tothwart replication of hyperproliferating cells, other cellularpopulations may be targeted as well. For example, cells infected by apathological agent such as a-virus or bacterium, may also be targeted.

Exposure of DNA to the agents of the present invention results inbinding of the agents to the DNA and subsequent cleavage of the DNA. Thecleavage pattern may be random. Alternatively, the complexes of thepresent invention may be purposefully designed to favor binding atparticular regions of the DNA and so affect site specific (or at leastsite-preferential) cleavage. For example, the complexes may be designedto bind preferentially to a particular sequence of bases, or to aparticular structural motif or location (e.g. to A-, B-, or Z-DNA, or tothe major or minor groove). It is also possible to append to thissupramolecular structure architecture recognition sites that would leadto site specific cleavage of DNA. For example, single stranded DNAsequences can be appended to the complexes to allow recognition ofcomplementary strands and subsequent selective cleavage at the site ofmetal complex attachment. Further, proteins or fragments of proteinsthat bind selectively to specific regions of a DNA molecule may also beattached, e.g. topoisomerases, gyrases, DNA polymerases, etc. Methods ofattaching or appending additional substituents to the complexes of thepresent invention would be well-known to those of skill in the art, e.g.by substitution of a non-essential ligand such as a terminal ligand. Thecomplexes may be used to cleave either double- or single-stranded DNA,as well as DNA-RNA hybrids, and double- or single-stranded RNA.

In preferred embodiments, the agents of the present invention bind toand cleave DNA within cells for which it is desired to attenuate theability to replicate. Without being bound by theory, the agents of thepresent invention appear to provide a less drastic mode of treatingpathological conditions which result from the hyperproliferation ofcells in that the agents appear to cause a cessation of replicationwithout killing the hyperproliferating cells outright. This is anadvantage because the immediate killing of, for example, all tumor cellsin a tumor mass can have unwanted results for a patient in which thetumor is being treated. If millions of tumor cells are killed outrightmany or most of the cells undergo lysis, releasing their contents intothe environment. The result of such a massive release of the contents ofdead cells into an area of the body can generate, for example,inflammatory and other unwanted reactions in otherwise healthy tissue inthe environment. By biasing the effects of the agent to a cessation ofreplication, the progression of the tumor is halted, and the tumor cellswill relatively gradually undergo cell death. Thus, the body of thepatient under treatment experiences less drastic treatment consequences.However, those of skill in the art will recognize that some cells in thehyperproliferating tissue may also be killed outright by exposure to theDNA cleaving agents of the present invention. Other potential benefitscould include attenuation of the cancer cells that would make them moresusceptible to other types of cell killing such as chemotherapy orradiotherapy. Indeed, the methods of the instant invention may bepracticed in conjunction with other such therapeutic measures.

The present invention provides specificity in attenuating cellularproliferation in that activation of DNA cleavage and subsequent celldamage and/or death will occur only when the cells containing thecleavage agent are exposed to suitable wavelengths of light. Suitablewavelengths of light for use in the practice of the present inventionare dependent on the components of a given supramolecular complex. Ingeneral, low energy, visible light is utilized. By “low energy, visiblelight” we mean light of wavelengths >475 nm. For example, the wavelengthused will depend on the complex of interest and its ability to absorb atthat wavelength as well as the ability of the wavelength of light topenetrate the applicable biological material. Typically excitation wouldoccur in the region of the intense metal to ligand charge transferexcitation. For example, for the system [{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅the lowest lying such MLCT transition center is at 514 nm so optimalexcitation would occur in this region (±about 50 nm) i.e. from about 464to about 564 nm. However, those of skill in the art will recognize thatother excitations further from the optimum can also be used due to theefficient internal conversion within supramolecular complexes of thetype described herein. For example, for the system[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ excitation is possible throughout the UVand into the visible region, i.e. from about 200 to 650 nm. Light for invivo applications where significant penetration is needed wouldtypically be in the therapeutic window of about 650 to about 950 nm.

Specificity also results in that, when the targeted cells are in vivo(i.e. located internally within an organism), they will be exposed tolight only when light of an appropriate wavelength is deliberatelyintroduced into the environment, for example, during a studied surgicalprocedure using, e.g., optical fibers. For endoscopic use, opticalfibers are threaded through a catheter or endoscope, allowing for smallincisions while delivering a focused beam of light. When the targetedcells are ex vivo, cells are shielded until light of the wavelength thatwould activate the photosensitizing agent could be purposefullyadministered. Many companies (such as Coherent Medical Group, CoherentInc., Palo Alto, Calif.), manufacture products specifically designed forthe production of narrow wavelengths of light required for medical use.Those of skill in the art are acquainted with and will recognize thatmany such products exist. For example, gas lasers as well as LEDs arecommercially available and capable of producing the requisite light. Anyappropriate means of illuminating the target cells that results inactivation of the photosensitizer molecule within the target cells, sothat injury or death of the target cells results, may be utilized in thepractice of the present invention. For example, of such methods ofillumination, see Bellnier, D. et al. 1999. Design and construction of alight-delivery system for photodynamic therapy. Med. Phys. 26: 1552.

Specificity may also be conferred by the attachment to the complex ofmoieties which serve to direct the complex to a desired target. Theagents may be coupled to targeting moieties such as antibodies, lectins,targeting fragments such as bacterial toxin molecules or fragments ofsuch molecules, all of which can serve to direct the cleaving agent tothe targeted population of cells, and also to promote uptake of thecomplex by the cell. For example, by coupling a DNA cleaving agent ofthe present invention to an antibody specific for an antigen that isexpressed on a particular type of tumor cell, the agent can be deliveredto the tumor cells of interest. See, for example, U.S. Pat. No.6,426,400 to Zalutsky (Jul. 30, 2002) and U.S. Pat. No. 6,492,123 toHollinger et al., (Dec. 10, 2002), the complete contents of which arehereby incorporated by reference.

Delivery of the DNA cleaving agents of the present invention to the DNAto be cleaved may be carried out by any of several known methods andwill vary from case to case, depending on the particular application.For example, for some laboratory applications, solutions of the agentsmay be mixed directly with the DNA to be cleaved. For the cleavage ofcultured cells (including ex vivo cells) the cleaving agents of thepresent invention may be added directly to the culture media where theyare taken up by the cells. For in vivo applications, those of skill inthe art will recognize that many means of administration exist,including but not limited to: direct application of the DNA cleavingagent in a suitable carrier, e.g. by topical administration to acancerous lesion such as a melanoma or other area of exposedhyperproliferating tissue; or by delivery directly into the tumor orother hyperproliferating tissue, e.g. by injection or other type ofdirect infusion. Other means of delivery include systemic delivery. Inthe case of systemic delivery, many cells will be exposed to andinternalize the agents of the present invention. However, only thosecells which are later exposed to suitable wavelengths of light will beeffected by the presence of the agent by cleavage of their DNA. Residualagent within non-targeted cells will be eliminated from the body over atime period of about two weeks, during which the patient must avoidexposure to wavelengths of light that would activate the agents.

Thus, the agents of the present invention may be administered by any ofseveral suitable means that are well-known to those of skill in the art.For example, intramuscularly, intravenously, intratumorally, orally(e.g. in liquid or tablet/capusular form), via suppositories, viainhalation, and the like.

In order to effect administration of the agents of the presentinvention, the present invention also provides a composition foradministration to hyperproliferating cells. The composition comprises atleast one of the DNA cleaving agents and a suitable carrier, e.g. asuitable physiological carrier for in vivo administration, e.g. saline.The composition may be administered in any of a variety of suitableforms, including forms that include additional components such asbuffers, stabilizers, nutrients, anti-oxidants, flavorings, colorants,and the like, which are appropriate to a means of administration. Thoseof skill in the art will recognize that the exact form will vary fromapplication to application. The compounds can be administered in thepure form or in a pharmaceutically acceptable formulation includingsuitable elixirs, binders, and the like or as pharmaceuticallyacceptable salts or other derivatives. It should be understood that thepharmaceutically acceptable formulations and salts include liquid andsolid materials conventionally utilized to prepare injectable dosageforms and solid dosage forms such as tablets and capsules. Water may beused for the preparation of injectable compositions which may alsoinclude conventional buffers and agents to render the injectablecomposition isotonic. Solid diluents and excipients include lactose,starch, conventional disintegrating agents, coatings and the like.Preservatives such as methyl paraben or benzalkium chloride may also beused. Depending on the formulation, it is expected that the activecomposition will consist of 1-99% of the composition and the vehicular“carrier” will constitute 1-99% of the composition.

Likewise, the dosage, frequency and liming of administration will varyfrom case to case and will depend on factors such as the particularapplication, the nature and stage of a condition resulting fromhyperproliferation of cells (e.g. size and location of a malignant ornon-malignant tumor), characteristics of the patient (e.g. overallhealth, age, weight, gender and the like), and other factors such asancillary treatments (chemotherapy, radiotherapy, and the like). Thedetails of administration are best determined by a skilled practitionersuch as a physician. Further, the details of administration are normallyworked out during clinical trials. However, the approximate dosage rangewill preferably be from about 0.1 to 10 mg of agent per kg of weight,and more preferably from about 0.25 to 1.0 mg/kg. When treating DNAdirectly, the amount of agent to be administered is preferably in therange of about 0.1-50 μg per about 0.1-50 μg of DNA, and morepreferably, in the range of about 1-10 μg per about 1-10 μg of DNA.Those of skill in the art will recognize that the precise amounts willvary depending, for example, on the precise characteristics of thecomplex and the DNA itself, on temperature, pH, and the like. Typically,the agent will be administered about 1 to 24 hours prior to exposure toa suitable light source, and preferably from about 1 to 4 hours prior toexposure to the light source.

Likewise, the dose or frequency of illumination of the target cells willvary from case to case, but will generally be in the range of 25-200J/cm² light dose, 25-200 mW/cm² fluence rate (see Ochsner, M. 1997.Photodynamic Therapy: the Clinical Perspective. Review on applicationsfor control of diverse tumours and non-tumour diseases. Drug Res.,47:1185-1194).

Further non-limiting embodiments of the invention are presented in thefollowing Examples section.

EXAMPLES Background for Examples 1 to 4

Interest in the area of supramolecular chemistry has resulted in thedesign of many photochemically and electrochemically activeruthenium(II) polypyridyl complexes.¹⁻¹⁷ Supramolecular complexes havebeen designed, taking advantage of the long-lived metal-to-ligand chargetransfer (MLCT) excited state of the widely studied [Ru(bpy)₃]²⁺chromophore,¹⁻³ focused on their use as photochemical moleculardevices⁴⁻⁹ (bpy) 2,2′-bipyridine. Incorporation of ruthenium(II)polypyridyl groups into a supramolecular motif eliminates the need formolecular collision resulting in facile electron or energy transfer. Thebridge, which links the metal centers in these supramolecular complexes,is often a multidentate polyazine ligand.⁴⁻¹⁷

Polymetallic complexes incorporating polyazine bridging ligands (BL)have received a great deal of attention.⁴⁻¹⁷ The BL serves to bring themetal centers into close proximity and creates a pathway for energy orelectron transfer. The commonly used bridging ligand2,3-bis(2-pyridyl)pyrazine (dpp) (FIG. 1A) binds to two metal centersthrough a pyridyl and a pyrazine nitrogen, acting as an AB chelate,resulting in a mixture of stereoisomers not typicallyseparated.^(4-9,12,14,15) Another BL which performs the same functionbut has not received as much attention is 2,2′-bipyrimidine (bpm) (FIG.1B), which binds to two metal centers through two equivalent nitrogenseliminating the stereoisomers associated with the ABchelates.^(11,13,16,17)

Within a supramolecular architecture, terminal ligands (TL), typicallybpy, are coordinated to the ruthenium light absorbers. Another TL usedin supramolecular complexes is 2,2′:6′,2″-terpyridine (tpy) (FIG. 1C).Although [Ru(tpy)₂]²⁺ has a short-lived excited state,¹⁸⁻²⁰ the tpyligand brings the advantage of eliminating the Δ and Λ isomeric mixturesassociated with the tris-bidentate metal centers giving somestereochemical control in supramolecular complexes. Long lived excitedstates are observed for many ruthenium tpy complexes incorporatingpolyazine bridging ligands.²¹⁻³⁰

Trimetallic complexes of the form [{(bpy)₂Ru(BL)}₂MCl₂]⁵⁺, where BL=dpp,2,3-bis(2-pyridyl)quinoxaline (dpq), and2,3-bis(2-pyridyl)benzoquinoxaline (dpb) and M=Ir(III),³¹⁻³³ have beenstudied, and a preliminary report of M=Rh(III) has appeared.^(32b) Thesystem with M=Ir and BL=dpb acts as a molecular device forphotoinitiated electron collection^(31a) and is an electrocatalyst forCO₂ reduction.³³ The bpm trimetallic complexes [{(bpy)₂Ru(bpm)}₂IrCl²]⁵⁺and [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ have Ru-(dπ) based highest occupiedmolecular orbitals (HOMOs) and bridging ligand, bpm(π*), based lowestunoccupied molecular orbitals (LUMOs).³⁴

A number of important studies on the coupling of ruthenium lightabsorbers to rhodium electron acceptors in suprammolecular frameworkshave appeared.^(32,34-44) Interesting systems with varying bridge lengthwere studied by Indelli, Scandola, Collin, Sauvage, and Sour,[(tpy)Ru(tpy(Ph)_(n)tpy) Rh(tpy)]⁵⁺ (n=0, 1, or 2).³⁶ Linked bpy systemsof the type[(Me₂phen)₂Ru^(II)(Mebpy-CH₂CH₂-Mebpy)Rh^(III)(Mebpy)₂]^(5+ 36,37) and adpp bridged system [(bpy)₂Ru^(II)(dpp)Rh^(III)(bpy)₂]⁵⁺ ³⁵ have beeninvestigated. Endicott et al. have studied Ru^(II), Rh^(III)cyanide-bridged complexes.⁴¹ Often these systems are reported to undergointramolecular electrontransfer quenching of the Ru-based MLCT excitedstate by the rhodium center.

A trimetallic structural motif would be an interesting framework toexploit the electron acceptor properties of the rhodium metal center.This requires the development of synthetic methods and the ability tomodulate orbital energies in a supramolecular architecture. Within thisframework the trimetallic complexes [{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃ and[{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃ have been synthesized and characterizedby FAB mass spectral analysis, electronic absorption spectroscopy,electrochemistry, and spectroelectrochemistry. These complexes coupletwo ruthenium light absorbers (LA) to a central electron collecting (EC)rhodium metal center to form a LA-BL-EC-BL-LA assembly. The interestingeffects of bridging ligand and terminal ligands on the spectroscopic andelectrochemical properties of these complexes is discussed.

Material and Methods for Examples 1 to 4

Materials.

2,2′:6′,6″-Terpyridine (tpy) (GFS chemicals), ruthenium(III) chloridehydrate, rhodium trichloride hydrate, and 2,2′-bipyrimidine (bpm)(Alfa), triethylamine (Acros), 2,3-bis(2-pyridyl)-pyrazine (dpp)(Aldrich), (80-200 mesh) adsorption alumina (Fisher), and spectroqualitygrade acetonitrile and toluene (Burdick and Jackson) were used asreceived. Tetrabutylammonium hexafluorophosphate Bu₄NPF₆ (used assupporting electrolyte for electrochemistry experiments) was prepared bythe aqueous metathesis of tetrabutylammonium bromide (Aldrich) withpotassium hexafluorophosphate (Aldrich). After severalrecrystallizations from ethanol the white crystals were dried undervacuum and stored in a vacuum desiccator. Elemental analysis wasperformed by Galbraith Laboratories, Inc., Knoxville, Tenn.

Synthesis.

(tpy)RuCl₃,⁴⁵ [(tpy)RuCl(dpp)](PF₆),⁴⁶ [(tpy)RuCl(bpm)](PF₆),⁴⁷[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅,^(32b) and [{(bpy)₂Ru(bpm)}₂RhCl₂](PF₆)₅³⁴ were synthesized as described previously.

[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃.

A solution of 0.40 g (0.54 mmol) of [(tpy)RuCl(dpp)](PF₆) and 0.080 g(0.36 mmol) of rhodium trichloride hydrate in 2:1 EtOH/H₂O was heated atreflux for 1 h. After being cooled to room temperature, the reactionmixture was added dropwise to an aqueous solution of 100 mL of H₂0 and100 mL of saturated KPF₆(aq) solution with stirring. The resultingprecipitate was filtered, washed with 30 mL of cold water and 30 mL ofcold ethanol followed by 30 mL of ether, and air-dried for 30 min. Theproduct was dissolved in a minimum amount of acetonitrile (ca. 5 mL),flash precipitated in 200 mL of ether, and collected by vacuumfiltration to yield a purple powder (0.40 g, 0.22 mmol, 82% yield).Anal. Calcd for [{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃, 8H₂O; C, 35.52; H, 2.98;N, 10.00. Found: C, 35.20; H, 2.35; N, 9.93. UV/vis (CH₃CN): λmax (nm)[ex 10⁻⁴ M⁻¹ cm⁻¹]) 274 [4.70], 314 [6.48], 360 (sh) [2.72], 460 [1.13],540 [2.72]. FAB-MS ion (m/z; relative abundance):[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₂ ⁺ (1673, 100);[{(tpy)RuCl(dpp)}₂RhCl](PF₆)₂ ⁺ (1636, 10);[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)⁺ (1527, 25); [{(tpy)RuCl(dpp)}₂RhCl](PF₆)⁺(1493, 10).

[{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃.

A solution of 0.32 g (0.49 mmol) of [(tpy)RuCl(bpm)](PF₆) and 0.070 g(0.32 mmol) of rhodium trichloride hydrate in 2:1 EtOH/H₂O was heated atreflux for 2 h. After the reaction mixture was cooled to roomtemperature, a black residue was removed by filtration. The filtrate wasadded dropwise to an aqueous solution of 100 mL of H₂0 and 1002 mL ofsaturated KPF₆(aq) solution with stirring. A brown precipitate formed,which was filtered and washed with 30 mL of cold ethanol followed by 30mL of ether. The resulting brown product was dissolved in a minimum ofacetonitrile (ca. 5 mL), flash precipitated in 200 mL of ether, andcollected by vacuum filtration to yield a greenish/brown powder (0.28 g,0.17 mmol, 72% yield). Anal. Calcd for [{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃,CH₃CN, H₂O; C, 33.45; H, 2.28; N, 12.19. Found: C, 33.33; H, 2.40; N,11.76. UV/vis (CH₃CN): λmax (nm) [ε×10⁻⁴ M⁻¹ cm⁻¹]) 272 [6.21], 312[5.50], 330 (sh) [3.11], 464 [2.50], 656 [1.00]. FAB-MS ion (m/z;relative abundance): [{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₂+(1520, 85);[{(tpy)RuCl(bpm)}₂RhCl](PF₆)₂ ⁺ (1485, 15);[{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)⁺ (1375, 100);[{(tpy)RuCl(dpp)}₂RhCl](PF₆)⁺ (1340, 20).

Electronic Spectroscopy.

Electronic absorption spectra were recorded at room temperature using aHewlett-Packard 8452 diode array spectrophotometer with 2 nm resolution.Samples were run at room temperature in Burdick and Jackson UV-gradeacetonitrile in 1 cm quartz cuvettes.

Electrochemistry.

Cyclic voltammograms were recorded using a one-compartmentthree-electrode cell, Bioanalytical Systems (BAS), equipped with aplatinum wire auxiliary electrode. The working electrode was a 1.9 mmdiameter glassy carbon disk from BAS. Potentials were referenced to aAg/AgCl electrode (0.29 V vs NHE), which was calibrated against theFeCp₂/FeCp₂ ⁺ redox couple (0.67 V vs NHE).48 The supporting electrolytewas 0.1 M Bu₄NPF₆, and the measurements were made in Burdick and JacksonUV-grade acetonitrile, which was dried over 3 Å molecular sieves.

Spectroelectrochemistry.

Spectroelectrochemical measurements were conducted according to apreviously described method using a locally constructed H-cell whichuses a quartz cuvette as the working compartment.49 The working andauxiliary compartments were separated by a fine porous glass frit. Theworking electrode and auxiliary electrodes were high surface areaplatinum mesh, and the reference electrode was Ag/AgCl (0.29 V vs NHE).The measurements were made in 0.1 M Bu₄NPF₆/acetonitrile solutions thatwere 2×10⁻⁵ M metal complex. The electrolysis potential was controlledby a BAS 100 W electrochemical analyzer.

FAB Mass Spectrometry.

FAB mass spectral analysis was performed by M-Scan Incorporated, WestChester, Pa., on a VG Analytical ZAB 2-SE high-field mass spectrometerusing m-nitrobenzyl alcohol as a matrix. The trimetallic gave very niceFABMS patterns with sequential loss of each PF₆ ion being observed. Thefragmentation pattern was consistent with the proposed molecularstructure.

Example 1 Synthesis

The supramolecular complexes [{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃ and[{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃ were prepared in good yields under mildconditions using a building-block approach. It is this method thatallows for easy variation of structural components within thisstructural motif. The tpy is first bound to ruthenium followed by BLattachment.^(45,46) The trimetallic complexes are assembled by reactionof the [(tpy)RuCl(BL)](PF₆), where BL=dpp or bpm, with a slight excessof rhodium(III) trichloride hydrate. The synthesis of[{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃ by this method is illustrated in FIG. 2.This method of binding the bpm or dpp ligand to the ruthenium metalcenter first and then binding to the rhodium metal center yields cleanreactions with easily purified products. The use of excess rhodium(III)trichloride hydrate ensures that most of the monometallic precursor isreacted. The major product in each case is the desired trimetallic. Theexcess rhodium(III) trichloride is easily removed by aqueous washings ofthe precipitated hexafluorophosphate salt of the trimetallic complex.

The use of dpp as a bridging ligand leads to cis and trans typestereoisomers, around the Ru which are not detectable by cyclicvoltammetry or electronic absorption spectroscopy.^(46,47) Utilizationof the symmetric bridging ligand bpm eliminates the cis/trans typestereoisomers present in the dpp synthons.

These trimetallic complexes were effectively characterized by FAB massspectral analysis. These supramolecular complexes typically show highmass peaks that are easy to interpret with loss of counterions andintact ligands. Fragmentation patterns for these trimetallics showsequential loss of PF₆ ⁻ counterions and the chlorides bound to therhodium center.

This example demonstrates that a method has been developed to preparesuch complexes that is general and allows for component modification andthat the described complexes have the proposed formulation.

Example 2 Electrochemistry

Trimetallic complexes of the form [{(bpy)₂Ru(CL)}₂RhCl₂]⁵⁺ arecharacterized by reversible ruthenium oxidations, irreversible rhodiumreductions, and reversible ligand reductions, with the BLs (dpp or bpm)being reduced prior to the bpy ligands.^(32,34) They display a Ru(dσ)HOMO. The LUMO is localized on Rh(dσ*) for dpp and bpm(π*) for the bpmbridged system.

The cyclic voltammogram of [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ in 0.4 MBu₄NPF₆/CH₃CN solution is illustrated in FIG. 4A and summarized in Table1.

TABLE 1 Electrochemical Properties for a Series of Ru(II) andRu(II)/Rh(III)/ Ru(II) Trimetallic Complexes Where tpy = 2,2′:6′,2″-Terpyridine, dpp = 2,Bis(2-pyridyl)pyrazine, and bpm = 2,2′-BipyrimidineE_(1/2) in V^(a) (ΔE_(p) in mV) assignment[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃ 1.12 (85) 2Ru^(III/II) E_(p) ^(c) = 0.47Rh^(III/I) −0.87 (140) dpp, dpp/dpp, dpp⁻ −1.20 (95)  dpp, dpp⁻/dpp⁻,dpp⁻ [{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃  1.12 (100) 2Ru^(III/II) E_(p) ^(c)= −0.26 Rh^(III/I) E_(p) ^(c) = −0.38 Rh^(II/I) −0.70 (100) bpm,bpm/bpm, bpm⁻ −1.12 (115) bpm, bpm⁻/bpm⁻, bpm⁻[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅-^(32b) 1.6 2Ru^(III/II) E_(p) ^(c) = −0.39Rh^(III/I) −0.79 dpp, dpp/dpp, dpp⁻ −1.02 dpp, dpp⁻/dpp⁻, dpp⁻[{(bpy)₂Ru(bpm)}₂RhCl₂](PF₆)₅ ³⁴ 1.7 2Ru^(III/II) −0.13 bpm, bpm/bpm,bpm⁻ −0.26 bpm, bpm⁻/bpm⁻, bpm⁻ −0.78 Rh^(III/I) [(tpy)RuCl(dpp)](PF₆)⁴⁷1 Ru^(III/II) −1.21 dpp^(0/—) −1.54 tpy^(0/—) [(tpy)RuCl(bpm)](PF₆)⁴⁷1.01 Ru^(III/II) −1.15 bpm^(0/—) −1.56 typ^(0/—) ^(a)Potentials reportedversus the Ag/AgCl (0.29 V vs NHE) reference electrode in 0.1 MBu₄NPF₆CH₃CN.

A reversible redox couple at 1.12 V is observed in the positivepotential region. This redox couple is attributed to two overlappingRu^(II/III) oxidations. These LAs are largely electronically uncoupled,allowing them to function independently.^(31,34) The Ru^(II/III) couplesoccur 480 mV less positive in the [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ systemsrelative to the bpy systems, resulting from the chloride coordination onthe Ru centers in the tpy systems. Reductively an irreversible peak isobserved at −0.47 V. This couple results from the overlapping reductionof the Rh(III) to Rh(II) and then to Rh(1). Similar behavior is reportedby DeArmond for the [Rh(bpy)₂Cl₂]⁺.⁵⁰ Reduction of the Rh(III) to Rh(I)should be followed by conversion of the formally d⁶ pseudocathedralRh(III) to a square planar d⁸ Rh(I). This occurs by chloride loss asevidenced by the presence of free chloride seen in anodic scans thatfollow cathodic scans through the Rh^(III/I) couple. No evidence ofRh(I) reoxidation is seen in multiple scan experiments. Twoquasi-reversible redox couples at −0.87 and −1.20 V are attributed tosequential reduction of the two equivalent dpp bridging ligands,dpp,dpp/dpp,dpp⁻ and dpp,dpp⁻/dpp⁻,dpp⁻, respectively. Further reductivescanning results in a neutral species leading to adsorption of thecomplex onto the electrode surface. [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ exhibitsa ruthenium(I) based HOMO and a rhodium(III) based LUMO, analogous to[{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺. The proposed electrochemical mechanism isshown in FIG. 5. The cyclic voltammogram of [{(tpy)RuCl(bpm)}₂RhCl₂]³⁺in 0.4 M Bu₄NPF₆/CH₃CN solution is illustrated in FIG. 4B and summarizedin Table 1. A single reversible oxidation wave is observed atE_(1/2)=1.21 V and is assigned to the two overlapping Ru^(II/III) redoxcouples, indicating that the two ruthenium centers are largelyelectronically uncoupled. Two closely spaced irreversible reductions at−0.26 and −0.38 V in FIG. 4B are assigned as sequential one-electronreductions of the rhodium center, Rh^(III/II) and Rh^(II/I).Interestingly, when bpm is used as the BL these two couples shift apartrelative to the dpp analogue, indicating some stability of the Rh(II)oxidation state. This is an unusual property for a [Rh(NN)₂Cl₂]′ system.Reversing the scan after the Rh^(III/II) couple but prior to the RhI^(I/I) couple does lead to the observation of a small return wavecorresponding to Rh(II) reoxidation, but this couple remains largelyirreversible. Further cathodic scanning past the Rh^(II/I) couplereveals the sequential one-electron reduction of the bpm bridgingligands, bpm,bpm/bpm,bpm⁻ and bpm,bpm⁻/bpm⁻,bpm⁻. Further reductionleads to adsorption.

The new bpm-based trimetallic complex [{(tpy)RuCl(bpm)}₂RhCl₂]³⁺displays a Rh(dσ*) LUMO in marked contrast to the bpm(π*) LUMO in[{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺. The redox chemistry of[{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ is characterized by two reversibleone-electron bpm⁻ based reductions at −0.13 and −0.26 V followed by theirreversible reduction of the rhodium center, Rh^(III/I), at −0.78 V.³⁴Variation of the terminal ligands on the Ru metals indirectly modulatesthe energy of the bpm ligand orbitals. Coordination of the Cl⁻ ligand toruthenium in [{(tpy)RuCl(bpm)}₂RhCl₂]³⁺ results in a more electron richRu center. This leads to less stabilization of the bpm(π*) orbitalsrelative to the bis-bpy analogue. As the bpm(π*) and Rh(dσ*) orbitalsare very close in energy, this modulation of the bpm(π*) orbitalenergies by terminal ligand variation leads to orbital inversion, FIGS.3A and 3B.

This electrochemical data indicates that, in the trimetallic complexes[{(tpy)RuCl(BL)}₂RhCl₂]³⁺ and [{(bpy)₂Ru(BL)₂RhCl₂]⁵⁺, the BL(M) andRh(dσ*) orbitals are close in energy. In all cases the HOMO is localizedon the Ru(dπ) orbitals. The localization of the LUMO can be modulated,being Rh(dσ*) in nature for [{(tpy)RuCl(BL)}₂RhCl₂]³⁺ (BL=dpp or bpm)and [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ and bpm(π*) in nature for[{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺.

This example demonstrates that the complexes display redox patternsconsistent with their formulation. Additionally, the systems possess thenecessary energetics to undergo the needed metal to ligand chargetransfer excitation followed by intramolecular electron transfer toproduce the desired reactive metal to metal charge transfer state.

Example 3 Electronic Absorption Spectroscopy

The electronic absorption spectral data in acetonitrile of the newtrimetallic complexes, [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ and[{(tpy)RuCl(bpm)}₂RhCl₂]³⁺, as well as their monometallic precursors andtrimetallic bpy analogues are assembled in Table 2. The UV regions ofthe spectra for all of these complexes show BL (dpp or bpm) and terminalligand (tpy or bpy) π to π* transitions with the BLs expected to showthe lowest lying π to π* bands.^(1,4-9,34,47,51) The visible regions ofthe spectra are dominated by overlapping Ru(d π) to BL(π*) and Ru(d π)to bpy or tpy(π*) charge transfer (CT) transitions with BL based bandsoccurring at lower energy.

TABLE 2 Electronic Absorption Spectroscopy for a Series of Ru(II) andRu(II)/Rh(III)/Ru(II) Trimetallic Complexes Where tpy =2,2′:6′,2″-Terpyridine, dpp = 2,3-Bis(2-pyridyl)pyrazine, and bpm =2,2′-Bipyrimidine^(a) λ_(max) (nm) ε × 10⁻⁴ (M⁻¹ cm⁻¹) assignments[{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃ 274 4.7 tpy(π→ π*) 314 6.48 tpy(π→π*)330(sh) 5.41 Ru(dπ)→ tpy(π*) CT 360(sh) 2.72 dpp(π→ π*) 460 1.13 Ru(dπ)→tpy(π*) CT 540 2.72 Ru(dπ)→ dpp(π*) CT [{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃272 6.21 tpy(π→ π*) 312 5.5 tpy(π→ π*) 330(sh) 3.11 Ru(dπ)→ tpy(π*) CTbpm (π→ π*) 464 2.5 Ru(dπ)→ tpy(π*) CT Ru(dπ)→ bpm(π*) CT 656 1 Ru(dπ)→tpy(π*) CT [{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ ^(b) 242 6.53 bpy(π→ π*) 2849.64 bpy(π→ π*) 344(sh) 2.87 dpp (π→ π*) 414 1.74 Ru(dπ)→ bpy(π*) CT 5142.01 Ru(dπ)→ dpp(π*) CT [{(bpy)₂Ru(bpm)}₂RhCl₂](PF₆)₅ ³⁴ 278 9 bpy(π→π*) 412 3.7 Ru(dπ)→ bpy(π*) CT Ru(dπ)→ bpm(π*) CT 594 0.99 Ru(dπ)→bpm(π*) CT [(tpy)RuCl(dpp)](PF₆)⁴⁷ 238 2.32 dpp(π→ π*) 276 2 tpy(π→ π*)314 2.91 tpy(π→ π*) 370 0.44 Ru(dπ)→ tpy(π*) CT 514 0.89 Ru(dπ)→ tpy(π*)CT [(tpy)RuCl(bpm)](PF₆)⁴⁷ 240 3.94 bpm (π→ π*) 266 2.92 tpy (π→ π*) 3163.31 tpy (π→ π*) 370 0.96 Ru(dπ)→ tpy(π*) CT 516 0.99 Ru(dπ)→ tpy(π*) CTRu(dπ)→ bpm(π*) CT ^(a)Absorption spectra taken in acetonitrile at roomtemperature. ^(b)Lowest energy CT transitions taken from ref 32b.

The electronic absorption spectra for [{(tpy)RuCl(dpp)}₂RhCl₂]³ and[{(tpy)RuCl(bpm)}₂RhCl₂]³⁺ in acetonitrile are characterized byhigh-energy tpy and BL (π to π*) transitions, with tpy bands at 274 nmand 314 nm. A shoulder observed at ca. 340 or 360 nm is attributed tothe BL (π to π*) transition for dpp and bpm, respectively.^(31b)Significant spectral differences between these two trimetallics becomesapparent when the visible regions of the spectra are compared. Thelowest energy transition at 540 nm for [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺, whichcontains the Ru(dπ) to dpp(π*) CT transition, is 116 nm higher in energythan the corresponding transition for the bpm analogue. This suggeststhat the impact of the rhodium coordination on the BL π* orbitals ismore dramatic for bpm than dpp, consistent with the electrochemicalbehavior.⁴⁷

A comparison of the electronic absorption spectra of the trimetallic,[{(tpy)RuCl(dpp)}₂RhCl₂]³⁺, and its monometallic precursor,[(tpy)RuCl(dpp)]⁺, reveals some interesting features. The UV regions ofthe spectra are virtually identical, consisting of dpp and tpy based πto π* transitions. As expected, these transitions are more intense forthe trimetallic complex, in keeping with its molecular structure.Coordination of two monometallic precursors, [(tpy)RuCl(dpp)]⁺, to therhodium metal center red shifts the Ru(dπ) to dpp(π*) CT transition from516 nm for the monometallic to 540 nm. This results from rhodiumcoordination stabilizing the dpp-(π*) orbitals of the trimetallic,consistent with the electrochemical behavior of the title trimetallic.The Ru(dπ) to dpp(π*) CT band at 540 nm in [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ isred shifted relative to 514 nm in [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺. This shiftis due to higher energy Ru(dπ) orbitals in [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺due to the coordinated chloride, also consistent with theelectrochemical data.

The UV regions of the spectra for the bpm monometallic,[(tpy)RuCl(bpm)]⁺, and the trimetallic, [{(tpy)RuCl(bpm)}₂RhCl₂]³⁺,complexes are very similar, with intense intraligand π to π* transitionsfrom bpm and tpy. Upon coordination of the monometallic to the rhodiummetal center, the Ru(dπ) to bpm(π*) CT transition at 516 nm red shiftsto 656 nm. This is the result of stabilization of the bpm(π*) orbitalsfrom coordination of the electron-withdrawing rhodium center. This 656nm Ru(d π) to bpm(π*) CT transition of the title trimetallic is redshifted relative to the 594 nm peak in the bpy analogue[{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺, consistent with the electrochemical data.

Both title trimetallics [{(tpy)RuCl(BL)}₂RhCl₂]³⁺ possess Ru(dπ) basedHOMOs and Rh(dσ*) LUMOs. Spectroscopically, no optical transition isseen representing this metalto-metal charge transfer (MMCT) excitation.This likely results from the high extinction coefficient for the lowestenergy Ru(dπ) to BL(π*) CT transition and the low overlap of the Ru(d π)and Rh(dσ*) orbitals leading to low intensity of the MMCT transition.Energetically, this MMCT state lies lower in energy than the opticallypopulated MLCT state. This should lead to the intramolecular electrontransfer to the Rh center in these complexes leading to quenching of theMLCT emission, discussed below.

This example demonstrates that these complexes are efficient lightabsorbers and that they undergo excitation into a metal to ligand chargetransfer state with a high extinction coefficient. Additionally, thisexample demonstrates that the energy of this excitation can be tuned bysimple component modification within this supramolecular architecture.

Example 4 Spectroelectrochemistry

Spectroelectrochemistry was used to study the electronic absorptionspectroscopy and cyclic voltammetry of the title trimetallics. Thespectroelectrochemistry of [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ and[{(tpy)RuCl(bpm)}₂RhCl₂]³⁺ is shown in FIGS. 6A and 6B. The two-electronoxidation of [{(tpy)RuCl(dpp)}₂RhCl₂]³⁺ is greater than 95% reversible.Electrolysis at 1.35 V, past the Ru^(III/II) a redox couple, shows aloss of the absorption band at 540 nm. This is consistent with itsassignment as a Ru(dπ) to dpp(π*) CT transition. The absorption band at314 nm and its lowest energy shoulder at 360 nm broaden and shift tolower energy upon oxidation of the ruthenium metal centers, consistentwith a ligand-based (π to π*) transition.³¹ A component (at ca. 330 nm)is lost upon oxidation of the ruthenium centers, consistent with ahigher energy Ru(dπ) to tpy(π*) CT transition occurring in this region.Similar behavior has been reported for the oxidation of an array ofRu(tpy) moieties.^(52,53) Reduction of the trimetallic complex wasirreversible due to reaction of the reduced rhodium center, consistentwith the Rh(dσ*) nature of the LUMO.

Very similar spectroelectrochemistry is observed for the bpm-bridgedtrimetallic, [{(tpy)RuCl(bpm)}₂RhCl₂]³⁺, FIG. 6B. Oxidation of theruthenium centers at 1.45 V is greater than 95% reversible. Thiselectrolysis leads to the loss of the absorption bands at ca. 330, 464,and 656 nm, consistent with their assignment as higher energy Ru(dπ) totpy(π*), Ru(dπ) to tpy(π*), and Ru(dπ) to bpm(π*) CT transitions,respectively. A broadening and red shift of the absorption band at 312nm and shoulder at ca. 340 nm is consistent with overlapping intraligandπ to π* transitions. Reduction was irreversible, consistent with aRh(dσ*) LUMO.

We were unable to detect any emission from the title trimetallics atroom temperature or 77 K in acetonitrile solutions. This may result fromthe weak response of the photomultiplier tube in the region in whichthese complexes are expected to emit, a low quantum yield for emission,or a quenching of the MLCT excited state by the expected intramolecularelectron transfer to the rhodium metal center. The[{(bpy)₂Ru(bpm)}₂RhCl2]⁵⁺ system displays an emission at 800 nm, 34supporting the role of intramolecular electrontransfer quenching of theMLCT excited state in the title trimetallics in quenching their MLCTemission.

This example demonstrates that the nature of the lowest lying transitionis metal to ligand charge transfer in character. Additionally, thisexample demonstrates the Rh-based nature of the LUMO allowing this Rhmetal to function as an electron acceptor within this structural motif.This established the metal to metal nature of the lowest lying excitedstate of these complexes.

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Background for Example 5

Recent emphasis has been placed on developing reagents capable ofcleaving DNA, applicable as structural probes and therapeutic agents,with many transition metal complexes being reported.¹⁻¹⁸ Photochemicalapproaches are of particular interest as they offer reaction control andcan be highly targeted.^(10-15,19) One popular approach involves thesensitization of molecular oxygen.^(5,7,8,20)

The development of photosensitizers that absorb low energy light, aretunable, and function in the absence of molecular oxygen is of interest.Oxygen independent systems function under conditions of low oxygencontent and often have a different mechanism of photocleavage.¹⁶ Aphotosensitizer which can be excited with low energy light can avoid thebase damage induced by UV light.^(21,22)

Rhodium and ruthenium complexes photocleave DNA. Photolysis at 310 nm ofrhodium(III) complexes of phi (9,10-phenanthrenequinone diimine) leadsto hydrogen abstraction from the 3′-carbon of deoxyribose, leading toDNA cleavage.²³ Cleavage selectivity can be modulated by ancillary²⁴ andactive²⁵ ligand variation or by tethering to DNA.²⁶⁻²⁹[Rh(phi)₂(phen)]³⁺ has recently been shown to stabilize duplex DNAinhibiting transcription.30 Rh₂(O₂CCH₃)₄L₂ (L=H₂O₁₄ or PPh₃ ³¹) hasexhibited the ability to photocleave DNA when irradiated in the presenceof electron acceptors. Studies have shown site specific oxidativecleavage of DNA using [Ru^(IV)(tpy)(bpy)O]²⁺ and[Ru^(III)(tpy)(bpy)OH]²⁺ (tpy=2,2′:6′,2″-terpyridine.^(32,33)Photoexcitation of ruthenium(II) (polypyridyl systems has resulted inoxidative damage to DNA in the presence of an electron acceptor³⁴⁻³⁶ andcleavage by oxygen sensitization.^(5,7,8) Rh(III) complexes intercalatedinto DNA serve as electron acceptors for excited Ru chromophores vialong-range electron transfer.^(37,38)

Trimetallic complexes coupling light absorbing ruthenium centers toreactive metal centers have been of interest. [{(bpy)₂Ru(BL)}₂MCl₂]⁵⁺(M=Rh or Ir and BL=2,3-bis(2-pyridyl)pyrazine (dpp)³⁹ or2,2′-bipyrimidine (bpm)⁴⁰) complexes, shown in FIG. 7 display quitevaried electrochemical properties and differing lowest lying excitedstates. They are good chromophores with the high energy region of theelectronic absorption spectra dominated by ligand based (π to π*)transitions. The visible region contains metal to ligand charge transfer(MLCT) transitions to both acceptor ligands with the BL transition beingthe lowest energy.

The electronic absorption spectra of these supramolecular complexes areshown in FIG. 8. All three complexes possess lowest lying Ru(d π) to BLCT bands that occur in the low energy visible region. For[{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ and [{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺, the Ru(dπ) todpp(π*) CT transition occurs at 525 nm. The Ru(dπ) to bpm(π to π*) CTtransition for [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ occurs at 594 nm. The Ir and Rhanalogues, [({(bpy)₂Ru(dpp)}₂MCl₂]⁵⁺, have spectroscopy that isvirtually identical owing to their similar supramolecular structure andthe dominance of the Ru light absorbers on the spectroscopic propertiesof these systems. The electrochemical properties vary with BL and M for[{(bpy)₂Ru(BL)}₂MCl₂]⁵⁺, summarized in Table 3. The complexes exhibit asingle reversible oxidation wave in the anodic region (1.56 and 1.70 Vvs Ag/AgCl) attributed to the overlapping Ru^(III/II) redox couple forthe two equivalent Ru centers. [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ exhibitsreversible bridging ligand reductions prior to reduction of the centralRh metal.⁴⁰ [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ undergoes an irreversible twoelectron reduction of the Rh(III) metal center prior to reduction of thedpp BL. This orbital inversion, FIG. 9, of the dpp(π*) and Rh(dσ*)orbitals, allows the Rh to function as an electron acceptor giving alowest lying, Ru to Rh metal to metal charge transfer (MMCT) excitedstate in this complex. It is this state we exploit for DNAphotocleavage.

TABLE 3 Electrochemical Properties for a Series of Ru(II) andRu(II)/Rh(III)/ Ru(II) Trimetallic Complexes where bpy =2,2′-Bipyridine, dpp = 2,3-Bis(2-pyridyl)pyrazine, and bpm =2,2′-Bipyrimidine complex Complex E_(1/2), V^(a) assignment[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ 1.6 2Ru^(III/II) −0.39^(b) Rh^(III/I)−0.79 dpp, dpp/dpp, dpp⁻ −1.02 dpp, dpp⁻/dpp⁻dpp⁻[{(bpy)₂Ru(bpm)}₂RhCl₂](PF6)₅ 1.7 2Ru^(III/II) −0.13 bpm, bpm/bpm, bpm−0.26 bpm, bpm⁻/bpm⁻, bpm⁻ −0.78 Rh^(III/I)[{(bpy)₂Ru(dpp)}₂IrCl₂](PF₆)₅ 1.56 2Ru^(III/II) −0.39 dpp, dpp/dpp, dpp⁻−0.54 dpp, dpp⁻/dpp dpp ^(a)Potentials reported versus the Ag/AgCl (0.29V vs NHE) reference electrode in 0.1 M Bu₄NPF₆,CH₃CN. ^(b)E_(p) ^(c)value.

Example 5 Photocleavage of DNA with Trimetallic Supramolecular Complexes

The lack of a Rh(dσ*) LUMO in the [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ systemallowed us to use this as a very similar supramolecular architecturecontrol system with a lowest lying MLCT state. The Ir analogue,[{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺, served as a spectroscopically matched systemwith a lowest lying MLCT state.

pUC18 and pBluescript were used to probe pbotocleavage of DNA by gelelectrophoresis.^(14,26,41,42) pUC18 plasmid is 2686 bp (Bayou Biolabs).Irradiation used a 1000 W xenon arc lamp, a water IR filter, and a 475nm cut off filter. Solutions were 3.5 μM in metal complex and 6.9 mM inphosphate buffer (pH=7) and allowed for ionic association of thecationic metal complexes with DNA. Dexoygenation was accomplished bybubbling with Ar for 30 min prior to the photolysis of the samples in anairtight cell blanketed with Ar.

FIG. 10 shows imaged ethidium bromide stained agarose gels that revealthat the excited state of [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ photocleaves DNA.Lane 1 (FIG. 10 a) shows the λ molecular weight standard. Lane 2 (FIG.10 a) indicates that pUC18 plasmid is found mostly as the supercoiledstate (form I) with a small amount of nicked, circular DNA (form II).When irradiated (λ_(irr)≧475 nm) for 10 min, the plasmid alone (lane 3)does not cleave.⁴² When incubated at 37° C. for 2 h in the presence ofthe monometallic precursor, [(bpy)₂Ru(dpp)]²⁺ (lane 4), or in thepresence of the trimetallic complex, [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ (lane 6),the plasmid DNA is not cleaved. When irradiated for 10 min in thepresence of the monometallic precursor (lane 5), no evidence for DNAcleavage is observed. In the absence of molecular oxygen when theplasmid is irradiated for 10 min (λ_(irr)>475 nm) in the presence of[{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺ at a 1:5 metal complex to base pair ratio(lane 7), conversion of the supercoiled DNA to the nicked form isobserved. FIG. 10 c, lane 5, shows a similar cleavage of pBluescriptplasmid using a narrow band excitation. These cleavage reactions arealso observed in the presence of molecular oxygen. The photocleavage ofDNA by [{(bpy)₂Ru(dpp)} 2RhCl₂]⁵⁺ but not the monometallic rutheniumsynthon illustrates the role of the supramolecular architecture,including Rh, on the desired photoreactivity. The cleavage productmigrates slightly slower through the gel than native nicked plasmid, andsimilar results have been observed by Turro.¹⁴

To explore the role of the Rh LUMO, resulting in an MMCT excited state,on the DNA photocleavage, the bpm analogue [{(bpy)₂Ru(bpm)}2RhCl₂]⁵⁺ andthe Ir analogue [{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺, which contain inaccessibleRh(dσ*) and Ir(dσ*) orbitals,^(39,40) were studied for their ability tophotocleave DNA. The Ir analogue has nearly identical electronicabsorption spectroscopy to that of the Rh complex. This allows it tofunction well as a control system possessing a lowest lying MLCT state.The results of this study are shown in FIG. 10 b. Lanes 1 and 2 (FIG. 10b) are the plasmid controls. Lanes 3 and 5 reveal that when the plasmidis incubated at 37° C. in the presence of [{(bpy)₂Ru(bpm)}₂RhCl₂]⁵⁺ or[{(bpy)₂Ru(dpp)}₂IrCl₂]⁵⁺, respectively, at a 1:5 metal complex to basepair ratio, no DNA cleavage occurs. Similar solutions irradiated(λ_(irr)≧475 nm) for 10 min (lanes 4 and 6), in the absence of molecularoxygen, also do not result in DNA cleavage. Similar studies in thepresence of oxygen also do not result in DNA cleavage.

These results indicate that our mixed-metal supramolecular complex,[{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺, is capable of DNA photocleavage and similarsystems without a Rh(dσ*) based LUMO do not display this behavior. Thisillustrates that our modifications of the coordination environment,yielding the desired orbital ordering, [{(bpy)₂Ru(dpp)}₂RhCl₂]⁵⁺,creates a system that photocleaves DNA via an MMCT excited state.Additionally, photocleavage can occur in the absence of molecularoxygen.

This study presents a new structural motif for DNA photocleavage agents,functioning from a previously unstudied excited state for thisapplication. A frank cleavage is observed consistent with reactivityarising from the photogenerated Rh(II) site. This supramoleculararchitecture allows for substitution of components to tune properties ofthese systems, allowing for the development of many new complexes thatshould display similar reactivity.

REFERENCES FOR EXAMPLE 5

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Example 6 Photoinduced Inhibition of Cell Replication with[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅

Photoinduced inhibition of Vero cell replication by the supramolecularcomplex [{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ was investigated. All experimentswere carried out with 0.5 mg/mL of [{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ in MEMmedia, and the results are shown in FIG. 11A. This cell line normallyreplicates once each 24 hours. The line with diamond symbols (⋄) showsthe growth of the Vero cells after irradiation for 10 minutes at λ>475nm. As can be seen, exposure to light alone did not impact thisreplication. The line with square symbols (□) shows the results obtainedwhen Vero cells are incubated in the dark with various concentrations of[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅. As can be seen, exposure to[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ alone (i.e. without illumination) did notimpact replication.

In marked contrast, photolysis at λ>475 nm after incubation with[{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ greatly inhibits cell replication, asevidenced by the line with circle symbols (∘).

This example demonstrates that the complex [{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅is not toxic to cells in the dark. It further demonstrates that thecomplex [{(bpy)₂Ru(dpp)}₂RhCl₂](PF₆)₅ is able to greatly inhibit thereplication of cells after exposure of cells with this complex to lowenergy visible light. Thus, the complexes of the present inventiondisplay photodynamic action leading to inhibition of cell replication.

Example 7 Concentration Dependence of Photoinduced Inhibition of CellReplication with [{(bpy)₂Ru(dpp)}₂RhCl₂]Cl₅

The concentration dependence of photoinduced inhibition of cellreplication by the supramolecular complex [{(bpy)₂Ru(dpp)}₂RhCl₂]Cl₅ isshown in FIG. 11B. The line with circular symbols shows the impact onVero cell growth of the incubation of the cells in the dark with theindicated concentrations of [{(bpy)₂Ru(dpp)}2RhCl₂]Cl₅. The line withsquare symbols shows the growth of Vero cells incubated with the sameconcentrations of [{(bpy)₂Ru(dpp)}₂RhCl₂]Cl₅, and subsequent irradiationfor 10 minutes at λ>475 nm. As can be seen, irradiation at λ>475 nmgreatly inhibits the ability of Vero cells to replicate. This is likelydue to photoinduced death of the irradiated cells.

This example demonstrates that the complex [{(bpy)₂Ru(dpp)}₂RhCl₂]Cl₅ isnot toxic to cells in the dark at a wide range of concentrations. Itfurther demonstrates that the complex [{(bpy)₂Ru(dpp)}₂RhCl₂]Cl₅ is ableto greatly inhibit the replication of cells after exposure of cells withthis complex to low energy visible light and demonstrates theconcentration needed for such action. Thus, the complexes of the presentinvention display photodynamic action leading to inhibition of cellreplication.

Example 8 Photocleavage of DNA with Various Supramolecular MetallicComplexes

The ability of several supramolecular complexes to photocleave DNA wasassayed and the results are shown in FIG. 12A-C. Details of theexperiments are given in the figure legends. The complexes utilized wereA, [{(bpy)₂Os(dpp)}₂RhCl₂](PF₆)₅; B, [{(tpy)RuCl(dpp)}₂RhCl₂](PF₆)₃; andC, [{(tpy)RuCl(bpm)}₂RhCl₂](PF₆)₃. As can be seen, each complexdisplayed the ability to efficiently cleave DNA upon activation by lowenergy, visible light.

This example demonstrates that many components as described herein canbe successfully incorporated into the generic supramolecular metalliccomplex of the present invention and result in the production of acomplex that successfully cleaves DNA.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

We claim:
 1. A method for cleaving DNA comprising the steps of:combining said DNA with a supramolecular complex to form a mixture, saidsupramolecular complex comprising: at least one metal to ligand chargetransfer (MLCT) light absorbing metal, at least one bridging π-acceptorligand, and an electron acceptor metal selected from the groupconsisting of rhodium(III), platinum(IV), cobalt(III), and iridium(III),wherein said supramolecular complex is present in sufficient quantity tobind to and cleave said DNA, and exposing said mixture to light orradiant energy sufficient to activate said supramolecular complex tocleave said DNA, wherein said step of exposing said mixture to light orradiant energy induces the production of a metal-to-metal chargetransfer state within said supramolecular complex, and wherein saidmetal-to-metal charge transfer state mediates the cleavage of said DNA.2. The method of claim 1 wherein said at least one metal to ligandcharge transfer (MLCT) light absorbing metal is selected from the groupconsisting of ruthenium(II), osmium(III), rhenium(I), iron(II) andplatinum(II).
 3. The method of claim 1, wherein said at least onebridging π-acceptor ligand is selected from the group consisting of2,3-bis(2-pyridyl)pyrazine; 2,2′-bipyridimidine;2,3-bis(2-pyridyl)quinoxaline; and 2,3,5,6,-tetrakis(2-pyridyl)pyrazine.4. The method of claim 1, wherein said supramolecular complex furthercomprises at least one terminal π-acceptor ligand.
 5. The method ofclaim 4, wherein said at least one terminal π-acceptor ligand isselected from the group consisting of 2,2′-bipyridine;2,2′:6′,2″-terpyridine; triphenylphosphine; and 2,2′-phenylpyridine anddiethylphenylphosphine.
 6. The method of claim 1, wherein said DNA is ina hyperproliferating cell.
 7. The method of claim 6, wherein saidhyperproliferating cell is selected from the group consisting ofleukemia cells, ovarian cancer cells, Burkitt's lymphoma cancer cells,breast cancer cells, gastric cancer cells, and testicular cancer cells.8. The method of claim 1, wherein said DNA is in a non-malignanthyperproliferating cell.
 9. The method of claim 8, wherein saidnon-malignant hyperproliferating cell is associated with psoriasis,warts or macular degeneration in an organism.